The Experimental Study Of Pathophysiological Mechanism Underlying Muscle-Eye-Brain Disease

Background:Muscle-Eye-Brain Disease is known to be an autosomal recessive genetic disease with defect in gene encoding protein-O-linked mannoseβ1,2 -N-acetylglucosaminyl -transferase1 (POMGnT1 ). Its clinical manifestations include severe muscular dystrophy, ocular defects and mental retardation . Many studies have been done to try to elucidate the underlying pathophysiological mechanism . However , some issues , for example , how abnormal neuronal migration occurs and what other changes in biological activites are happening still remain unsolved.Purpose:To explore the pathophysiological mechanism of Muscle-Eye-Brain Disease , understand the process of neuron migration defect and the gene expression profiles .Methods:1.Generation of POMGnT-1 knockout mice and genotyping of mice with wildtype as control.2.Immunofluorescence staining was performed for markers of the basement membrane, glia limitans, and granule neuron development ; double immunofluorescence staining was performed to manifest the relationship between laminin and GFAP ; Golgi staining was performed to show the morphology of neurons in cerebellum.3. BrdU was injected subcutaneously and immunostaining of BrdU was used to identify the neurons in S-stage for birthdating . 4. Electron microscopic analysis was conducted to examine the integrity of the pial basement membrane and granule neuron differentiation.5.YFPH transgenetic mice were used to show mossy fibers labeled by yellow fluorescence protein.6.Dissect cerebellar cortex and extract mRNA .7.Affymetrix genechip and microarray analysis8.production of cDNA and validation of microarray dataResults:1. Localized breaches in pial basement membrane and disruptions in the glia limitans were strongly associated with ectopia of EGL cells. In such ectopias, Bergmann glia fibers were retracted, disorganized, or protruded into the area. Thus, migration failure was correlated with the lack of normal Bergmann glia scaffold. Nevertheless, the ectopic EGL cells developed into mature granule neurons and formed synapses with mossy fibers.2. The expression levels of many genes in POMGnT-1 deficient mutant mice have increased compared with the heterozygotes mice .Among all these changed genes ,the expression levels of S100 calcium binding protein A5 , tyrosine hydroxylase , zinc finger protein of the cerebellum 1,calbindin 2 , guanine nucleotide binding protein (G protein), gamma 4 subunit have increased with higher folds (27.39 , 13.97,13.94 ,11.64 ,11.00 respectively). The expression levels of only a few genes ( neurofilament3 ,medium , POMGnT-1 and one gene encoding an unknown protein) have decreased. The changed genes have been categorized into several functional groups and among these groups transcription , metabolism, developmemt and signal transduction groups have more changed genes compared with other groups. Conclusion:1. Pial basement membrane breaches and glia limitans disruptions are the underlying causes of cerebellar granule neuron ectopia in POMGnT1 knockout mice and that migration into the IGL is not required for their differentiation.2. The gene expression profiling of POMGnT-1 mice provides some clues for further study of the pathophysiological mechanism of muscle -eye-brain disease.